An excellent correlation exists between the production of proteases by cells and their invasive behaivor in both normal (e.g., angiogenesis) and pathological (e.g., metastasis) processes. Serine-, cysteine-, and metallo-proteases have all been shown to be involved. This has led to the hypothesis that proteases play an obligatory role in cell invasion by degrading and allowing penetration of the extracellular matrix. Further support for this hypothesis has come from the observation that cartilage, which is resistant to tumor cell metastasis, and which blocks angiogenesis in vitro, is rich in inhibitors of serine-, cysteine-, and metallo-proteases. Cartilage depleted of those inhibitors by extraction is easily invaded by tumor cell and no longer blocks angiogenesis. Direct proof that protease production is essential for cellular invasion, however, awaits experiments in which purified inhibitors are shown to block cellular invasion in model systems. We propose to purify to homogeneity and characterize biochemically and enzymatically cysteine protease inhibitors from bovine cartilage and human amniotic fluid. Antibodies raised against the inhibitors will be used for immunofluorescence and immunoprecipitation experiments to determine their location and site of synthesis in a number of tissues and cultured cells. The purified inhibitors will also be tested alone, and in combination with purified collagenase and serine protease inhibitors, for their ability to block angiogenesis and tumor cell invasion in vitro, in defined experimental systems. If positive results are obtained in vitro, we shall use the same inhibitor(s) in in vivo assays of angiogenesis and metastasis in the future. The proposed research is relevant to the understanding of several areas of biology and medicine. Angiogenesis occurs throughout normal development. Other tissue remodelling during development may also require controlled proteolytic activity. In addition, cancer, arthritis, diabetic retinopathy, and other pathologies all involve cellular invasiveness. If the invasiveness of cells causing the pathologies can be halted by the use of purified protease inhibitors, the specific targeting of these inhibitors to the abnormal cells may have wide-ranging therapeutic implications.